Project Summary Blood donors are a genetically diverse population with numerous biological variables including sex, race, and age that may affect red blood cell (RBC) storage and transfusion outcomes. We have demonstrated a sex dichotomy in RBC predisposition to hemolysis, for which male RBCs from humans or mice exhibit enhanced susceptibility to cold storage, osmotic shock, and oxidative stress. A key discovery of our studies is that orchiectomy or testosterone replacement therapy (TRT) in mice significantly modulates RBC predisposition to hemolysis in storage and after transfusion. This finding has alerted us to the potential risks associated with TRT in blood donors. Testosterone therapies have been recently identified as of one of the most overused medical practices in the US, and the lack of clear guidelines and risk assessment of RBC transfusion from TRT patients deem such donors eligible for donation, except for cases of suspected polycythemia. Our preliminary genome-wide association studies in 25 mouse strains suggested that sex differences in predisposition to hemolysis involve gene networks surrounding p38 MAPK, which may intensify hemolytic response during stress, and can be activated by testosterone. These observations inform our overarching hypothesis that testosterone modulates erythroid cell differentiation and biology leading to sex differences in RBC characteristics and kinase activity under hemolytic stress including cold storage. In the current proposal, we use innovative human and mouse studies to evaluate the impact of donor sex and testosterone on transfusion outcomes and to map out down-stream p38 MAPK signaling pathways that modulate RBC structure, function and integrity during storage and transfusion. In Aim 1, we will define the impact of TRT in blood donors on RBC storage and post transfusion recovery using a human to mouse transfusion model. In Aim 2, we will determine the molecular interactions between testosterone and p38 MAPK, and identify the p38 MAPK signaling hubs that impact RBC function and survival in storage. The impact of testosterone on p38 MAPK biology (Aim 2A) will be defined at different levels of erythroid cell differentiation using human erythroid cell lines; in mature RBCs from TRT patients before and 120 days after testosterone treatments; and in RBCs from mice expressing attenuated p38 MAPK activity (B6.Cg-Mapk14tm1.1Dvb/J). In all experiments, we will monitor for changes in protein expression downstream of p38 MAPK including MK2, HSP 27 and anion exchangr-1. Next, we will use mouse and donor (NHLBI RBC-Omics) genome-wide association (GWA) databases of sex differences in hemolysis to enhance discovery of signaling hubs associated with p38 MAPK and regulated by sex. Outcomes from this project are likely to advance the field of transfusion medicine by assessing the risks associated with transfusion of blood from TRT donors; providing new insights into the mechanisms that mediate sex differences in RBC storage stability; and identifying signaling hubs that can be targeted therapeutically to reduce the risk of hemolysis in transfusion practices.